Maintenance of genomic stability requires accurate DNA replication. Eukaryotic cells achieve this by licensing the replication origins through recruiting the licensing factors to the origins to initiate replication and subsequently by 26S proteasome-mediated degradation of the licensing actors to prevent DNA re-replication. Eukaryotes must also maintain multiple copies of an extranuclear genome, the mitochondrial DNA (mtDNA), but the underlying mechanism for maintenance of mtDNA copy number remains a mystery. The current proposal is aimed at understanding the molecular mechanism of mtDNA replication licensing and is built on our recent discovery of HslVU protease as an essential regulator of mtDNA replication in Trypanosoma brucei, a protozoan parasite that contains an unusual mtDNA network, known as the kinetoplast DNA (kDNA). This protease is the first known regulator of kDNA replication and first bacterial-like HslVU protease identified in a eukaryote, but how it exerts its function is poorly understood, mainly because its regulatory proteins are not identified. Through tandem affinity purification, we identified two novel mitochondrial proteins that associate with TbHslVU in vivo, and we propose in this application to examine their potential roles in regulating TbHslVU as well as their potential involvement in kDNA replication. We hypothesize that the two novel proteins, named VUP1 and VUP2 for TbHslVU Partner 1 and 2, function as regulators of TbHslVU. They could either activate or inhibit the activity of TbHslVU or regulate the assembly of TbHslVU complex or mediate substrate recognition. The identification of regulatory proteins of TbHslVU suggests an additional level of control over the replication of kDNA and the complexity of the regulatory scheme of kDNA replication in trypanosomes. Most importantly, since no homologs of TbHslVU and its partners, VUP1 and VUP2, are found in humans, they are potential drug targets for anti-trypanosomiasis chemotherapy.